The human brain is comprised of billions of diverse neuronal types. How this diversity is generated and how these neurons are assembled into functional networks remain highly researched questions with unclear answers. Obtaining a deep understanding of how this is achieved promises to allow us to study the functions of different neuronal types (e.g., by gaining genetic access to them) as well as open the door to program stem cells into specific neuronal types to study and treat neurological diseases. Work spanning the last few decades in model organisms, such as Drosophila and mouse, has begun to unravel the underlying molecular mechanisms that lead to the specification of different neuronal types. At the very top of this molecular hierarchy are spatial and temporal programs that allow stem cells and their progeny to know where and when they are in space and time. For example, adult cholinergic neurons located in the basal forebrain (BF) and striatum are all born from a specific embryonic domain in the ventral telencephalon called the medial ganglionic eminence (MGE), which also produces precursors for other neuronal types such as GABAergic neurons. In addition to being spatially restricted, cholinergic neuronal types are born in overlapping temporal windows from E10-E13. Thus, the combination of spatial (MGE restricted) and temporal (E10-E13 restricted) programs contribute to cholinergic specification. Preliminary work in the Fishell lab has uncovered at least 8 distinct cholinergic neuronal types located in the BF and striatum but how these subtypes are specified during development is not known. As the specification into different cholinergic neuronal types likely occurs in the MGE upon becoming postmitotic (as is the case for GABAergic neurons), the goal of this proposal is to determine these specification programs. Understanding how different cholinergic neuronal types are specified will allow us to begin to understand their functions. Indeed, cholinergic neurons in the brain modulate neurocognitive functions such as memory, attention, and reward by regulating diverse brain circuits. Dysfunction of these neurons is linked to many neurological disorders, including Parkinson's and Alzheimer's diseases. In Aim 1, I will annotate the 8 adult (P30) cholinergic neuronal clusters, which I hypothesize represent the 2 interneuron types residing in different parts of the striatum and projection neurons targeting distinct brain areas. In Aim 2, I will define the developmental programs leading to different cholinergic classes by collecting and analyzing cholinergic precursors from E10-E13, which I will annotate by working backwards in time from our P30 dataset. In Aim 3, I plan to use existing methods and develop new strategies to assess the function of candidate factors in specifying cholinergic fates. The outcomes of these manipulations will be determined by charactering the expression of cluster specific markers, projection patterns, and changes in tra...